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Nosema ceranae infects honey bees (Apis mellifera) and
contaminates honey in Australia
Thomas Giersch, Tracey Berg, Francesca Galea, Michael Hornitzky
To cite this version:
DOI:10.1051/apido/2008065
Original article
Nosema ceranae infects honey bees (Apis mellifera)
and contaminates honey in Australia
*
Thomas G
iersch
, Tracey B
erg
, Francesca G
alea
, Michael H
ornitzky
Elizabeth Macarthur Agricultural Institute, New South Wales Department of Primary Industries, Private Mail Bag, 8, Camden, New South Wales, 2570, Australia
Received 3 July 2008 – Revised 1 October 2008 – Accepted 14 October 2008
Abstract – Polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis
and microscopy were used to test 307 adult bee and 37 honey samples collected in Australia for the pres-ence of two microsporidia, Nosema ceranae and Nosema apis. N. ceranae was detected in samples from 4 states (Queensland, New South Wales, Victoria and South Australia) and was most commonly found in samples from Queensland where 28 (33.7%) of 83 samples were positive. New South Wales had the second highest prevalence with 15 (15.8%) of 95 samples positive. South Australia and Victoria had 4 (16%) of 25 and 2 (4.5%) of 44 samples positive respectively. N. ceranae was not detected in samples from Western Australia and Tasmania. N. apis was detected in samples from all states. Three honey samples (8.1%) were PCR positive for N. ceranae. These positive honey samples originated from beekeepers in Queensland. Six imported honey samples tested were negative for both Nosema spp.
Nosema ceranae/ Nosema apis / nosemosis / Apis mellifera / PCR / RFLP
1. INTRODUCTION
Nosemosis is the most widespread of adult bee diseases and causes significant economic losses to beekeepers worldwide. This disease was originally thought to be caused by a sin-gle Nosema sp., Nosema apis Zander, a mi-crosporidian which has a range of effects on honey bee colonies and adult bees. The ef-fects of N. apis on the colony include spring dwindling of adult bee populations, decreased honey production, decreased brood produc-tion and in severe cases nosemosis may kill colonies (Langridge, 1961; Bailey and Ball,
1991; Fries,1995). N. apis reduces the lifes-pan of infected bees by about half in colonies in spring and summer (Kang et al.,1976). In-fected bees do not fully develop their hypopha-ryngeal glands, resulting in up to 15% of eggs
Corresponding author: M. Hornitzky michael.hornitzky@dpi.nsw.gov.au * Manuscript editor: David Tarpy
in severely infected colonies failing to pro-duce mature larvae in early summer (Wang and Moeller,1969). Infected queens are gen-erally superseded within a few weeks (Bailey and Ball,1991; Fries,1995).
In 1994, a microsporidian similar to N. apis was described in Asian honey bees (Apis
cer-ana Fabricius) from China (Fries et al.,1996). This parasite, called Nosema ceranae was sub-sequently detected in European honey bees (Apis mellifera L.) in Taiwan (Huang et al.,
2005). More recently it has been found in Brazil, USA, Vietnam, and much of Europe (Klee et al.,2007).
Whilst N. apis infection causes a fast act-ing, short duration syndrome, this has not been the case for N. ceranae, which instead has been observed in association with non-specific symptoms, such as a gradual depopulation, higher autumn/winter colony deaths or low honey production (Fries et al., 2006). It has also recently been shown that N. ceranae does not display the seasonality that is seen with
118 T. Giersch et al.
N. apis. In a study of bee samples collected in
Spain from 1999 to 2005 (Martin-Hernandez et al., 2007), the typical Nosema seasonal-ity was observed between 1999 and 2002, as characterised by an increase in infection levels especially in spring. However, from 2003 to 2005, this seasonality diminished and consis-tently high numbers of samples infected with
N. ceranae were detected throughout 2005
(Martin-Hernandez et al., 2007). N. ceranae has also been demonstrated to cause signifi-cantly higher mortalities in laboratory exper-iments indicating that it may be more virulent than N. apis (Paxton et al.,2007).
It is now clear that N. ceranae is not a new parasite of the European honey bee. N.
cer-anae was detected in bee samples collected in
the USA in 1996 and France in 2002 (Chen et al.,2008; Chauzat et al.,2007). The delay in recognising N. ceranae is attributable to the routine use of microscopy as a diagnostic tech-nique for the detection of Nosema-like spores. However, the spores of N. ceranae and N.
apis are similar in size, which has resulted in
their being diagnosed as the latter. Although, on average, N. ceranae spores are slightly smaller (Fries et al., 2006), microscopy can-not be used to reliably discriminate between the two species. Molecular techniques such as PCR have not been commonly used to di-agnose nosemosis, but these are necessary to identify each of the two species (Chen et al.,
2008; Martin-Hernandez et al.,2007). The ad-vent of molecular assays for Nosema spp. has driven the detection of N. ceranae worldwide. During a metagenomic study of microbes in honey bee colony collapse disorder (CCD) N.
ceranae was identified in a single sample of
apparently healthy bees imported from Aus-tralia while this study was being undertaken. Cox-Foster et al. (2007) detected N. ceranae in 30 (100%) of samples from colonies with CCD and 17 (80.9%) of non-CCD samples, suggesting a potential role for N. ceranae in CCD. Beyond the detection of N. ceranae in the single bee sample that had originated in Australia, nothing more is known about the presence of this microsporidian in Aus-tralia. This study was undertaken to determine whether bees in Australia are infected with
N. ceranae.
Australia is an island continent with restric-tions on the importation of beekeeping equip-ment. Honey, however, can be imported with-out restriction into all states except Western Australia and may be a reservoir for infection although N. ceranae has not been reported to be found in honey. This study also aimed to de-termine whether N. ceranae could be detected in honey.
2. MATERIALS AND METHODS 2.1. Collection and processing of adult
bee samples
Adult bee samples were collected by beekeepers and apiary inspectors in New South Wales, Queens-land, Victoria, South Australia, Western Australia and Tasmania. Submitters were asked to collect bees from the top lids of bee hives or from around the outside of the brood nest. Bee samples were stored in 70% (v/v) ethanol at room temperature prior to testing. These samples were collected from August 2007 to May 2008.
Fifteen adult bees from each sample were mac-erated in 15 mL of distilled water with a mor-tar and pestle. The suspension was decanted to re-move coarse material and examined by dark-field microscopy for the presence of Nosema spores. The homogenates were used directly for DNA extraction or stored at –20◦C until further use.
2.2. Collection and processing of honey samples
Honey samples were submitted by beekeepers and a honey packing plant. Six imported honey samples (Argentina, Spain, Korea [2] and Brazil [2]) were also examined for N. ceranae.
Honey samples (50 mL) were mixed with an equal volume of phosphate buffered saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4,
0.3 g/L KH2PO4) and centrifuged for 45 min at
Figure 1. (a) PCR products of N. apis and N. ceranae: Lane 1: O’Generuler DNA ladder; lane 2: N. ceranae
(252 bp); lane 3: N. apis (240 bp). (b) Lane 1, O’Generuler low range DNA ladder; lane 2, N. ceranae digested with DpnII (174, 78 bp); lane 3, N. apis digested with DpnII (119, 71, 50 bp).
2.3. Extraction of DNA from adult bees and honey
Bee homogenate (1 mL) or resuspended honey pellet (0.5 mL) was centrifuged for 15 min at 18 000 × g. The supernatant was discarded and DNA was extracted from the pellet with a DNeasy Plant Mini Kit (Qiagen) according to the manufac-turer’s protocol. The DNA was eluted in a final vol-ume of 50 µL and the extracts were stored at –20◦C until used as template in PCR.
2.4. PCR
The primers used in this study: NOS-FOR: 5’-TGCCGACGACGATGTGATATGAG-3’/ NOS-REV: 5’-CACAGCATCCATTGAAAACG-3’ (Higes et al., 2006) bind to the 16S rRNA gene of Nosema spp. to amplify products of 240 bp from N. apis and 252 bp from N. ceranae. Primer NOS-REV has 100% homology to both N. apis and
N. ceranae, whereas NOS-FOR is mismatched at 2
positions to N. ceranae (GenBank entry U26533, and all other available sequences); the 3’ AG in
N. apis (shown in bold) corresponds to GA in N. ceranae.
PCR was performed using a Mastercycler (Ep-pendorf) in a reaction volume of 20 µL con-taining 2 µL of template DNA, 1× PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.2 µM
of each forward and reverse primer and 0.7 U Taq polymerase (Roche). Given the mismatches be-tween NOS-FOR and N. ceranae, the PCR anneal-ing temperature was reduced from 62◦C to 55◦C to allow reliable amplification from both N. apis and
N. ceranae templates. Cycling conditions were as
follows: initial denaturation at 94◦C for 3 min, fol-lowed by 35 cycles of 20 s at 94◦C, 20 s at 55◦C and 30 s at 72◦C, and a final extension step at 72◦C for 5 min. PCR products were analysed on 2% (w/v) agarose gels. The O’Generuler low range DNA lad-der (Fermentas) was co-electrophoresed as a size marker. Gels were stained with ethidium bromide and visualised using UV illumination.
2.5. RFLP
Since the size difference of the PCR amplicons did not readily provide unambiguous discrimina-tion between the species N. apis and N. ceranae (Fig.1a), the sequences of N. apis (GenBank en-tries U26534, U97105, X73894, DQ235446), and
N. ceranae (Accession No. U26533) were aligned
using Sequencher (V4.7, Genecodes Corp.) and ex-amined for discriminating restriction endonuclease sites. The presence of a unique DpnII restriction site in all known N. apis sequences provided character-istic RFLP patterns for N. apis and N. ceranae.
The PCR amplicons were digested in a final vol-ume of 22.5 µL containing 2.0 U of DpnII (New England Biolabs) in 1× DpnII buffer , with incuba-tion at 37◦C for 18 h. Restriction fragments were separated in 3% (w/v) agarose gels and analysed as described above.
120 T. Giersch et al.
Table I. Prevalence of N. ceranae and N. apis in adult bee samples collected in Australia.
State of Origin N. ceranae N. apis Negative Total
Queensland 28 4 51 83
New South Wales 15* 48 34 95
Victoria 2 40 2 44
Western Australia 0 23 1 24
South Australia 4 4 17 25
Tasmania 0 23 13 36
TOTAL 49 142 118 307
* Two samples contained both N. ceranae and N. apis.
Table II. PCR results in relation to Nosema spore presence in adult bee samples
Microscopic analysis for PCR results
Nosema – like spores N. ceranae N. apis Negative Total
Negative 8 30 111 149
Positive 41* 112 7 158
TOTAL 49 142 118 307
* Two samples contained both N. ceranae and N. apis.
50 bp, whereas DpnII restriction of the 252 bp N.
ceranae amplicon yielded fragments of 174 and
78 bp.
3. RESULTS
3.1. Adult bee samples
Three hundred and seven adult bee samples were submitted for examination (Tab.I). These samples were primarily from individual bee-keepers from widely dispersed geographical regions within each state, except for 50 sam-ples from Queensland which were from one beekeeper and 16 samples which were from a single beekeeper from Tasmania.
PCR was demonstrated to be a more sensi-tive assay than microscopy for the detection of
Nosema (Tab.II). Of the 144 bee samples that were negative for Nosema-like spores when analysed by microscopy, 38 adult bee samples (26%), were found to be positive for Nosema spp when subjected to PCR-RFLP analysis (8 samples containing N. ceranae and 30 ples containing N. apis). Of the 163 sam-ples in which microscopy did detect Nosema-like spores, 7 samples (4%) were negative by PCR (Tab.II). Using microscopy Nosema-like
spores were not detected in the honey samples that tested positive for N. ceranae by PCR.
N. ceranae was detected in samples from 4
states (Queensland, New South Wales, Victo-ria and South Australia) and was most com-monly found in samples from Queensland where 28 (33.7%) of 83 samples were positive (10 of the 50 samples submitted by one sin-gle beekeeper from Queensland were N.
cer-anae positive). New South Wales had the
sec-ond highest prevalence with 15 (15.8%) of 95 samples positive. South Australia and Vic-toria had 4 (16%) and 2 (4.5%) of samples positive (Tab.I). N. ceranae was not detected in any samples from Western Australia and Tasmania.
A better assessment of the prevalence of N.
ceranae in each state and the relative
preva-lence of N. apis can be provided by presenting the number of N. ceranae-positive samples as a percentage of the number of Nosema posi-tive samples.Among the 32 bee samples from Queensland that were positive for N. ceranae or N. apis, 28 (87.5%) were found to carry N.
ceranae. For New South Wales, South
Aus-tralia and Victoria the percentage of N.
cer-anae positive samples were 23.8% (15
3.2. Honey samples
Thirty seven honey samples were tested during this study. Eight (21.6%) were PCR positive for N. apis and 3 (8.1%) were PCR positive for N. ceranae. The N. ceranae pos-itive honey samples originated from beekeep-ers in Queensland. All 6 of the imported honey samples were negative for both Nosema spp.
4. DISCUSSION
The combination of PCR and RFLP used in this study was demonstrated to be an ef-fective method of detecting N. ceranae and N.
apis. This methodology detected N. apis or N. ceranae in 38 samples which were found to
be negative using microscopy. The 7 bee sam-ples which tested negative using PCR-RFLP, but were positive using microscopy, contained very few Nosema-like spores. The detection of Nosema using PCR is also not restricted to the detection of DNA in Nosema spores. Hence, bees infected with DNA which may be contained in the polar filament extruded from spores or contained in cells in which spores have not yet developed may also be PCR pos-itive.
This study has demonstrated that in Aus-tralia N. ceranae infects bees in Queensland, New South Wales, South Australia and Victo-ria. However, there is a striking difference in the percentage of N. ceranae positive samples among the Nosema positive samples between states. Of the Nosema positive samples from Queensland 87.5% were N. ceranae positive but only 4.8% were positive from Victoria.
The four South Australian samples found to contain N. ceranae consisted of queen bees and escorts (worker bees caged with the queen) which had been imported from New South Wales. This indicates that queens and their escorts may be a source of infection al-though no other detections of N. ceranae were made from the remaining 21 samples in that state. These four samples should be considered as samples from New South Wales rather than South Australia.
The high percentage of N. ceranae-positive samples from Queensland compared with
those detected from New South Wales and Victoria may be due to two reasons. N.
cer-anae may have been first introduced into
Aus-tralia in Queensland and thus been established longest in that state. Most commercial bee-keepers in Australia are migratory, often mov-ing from state to state to take advantage of honey flows and suitable conditions for their bees. In doing so, N. ceranae may have spread to other colonies through movement of in-fected bees into and out of Queensland, or the sale of N. ceranae-contaminated equipment. Alternatively, N. ceranae may have been in-troduced elsewhere in Australia, but be better adapted to multiply and disseminate in tropi-cal climates such those occurring in Queens-land. Although N. ceranae has been reported in much of Europe, the impact of N. ceranae in Spain seems to be greater than in other countries with more temperate conditions in Europe (Martin-Hernandez et al., 2007). The same may hold true for the more temperate cli-mates of New South Wales and Victoria.
N. ceranae was detected far more
com-monly than N. apis in samples from Queens-land than in samples from other states. Of the Nosema positive samples from Queens-land 28/32 (87.5%) contained N. ceranae and 4/32 (12.5%) contained N. apis (Tab.I) indi-cating that N. ceranae may be replacing N.
apis. The replacement of N. apis with N. cer-anae has previously been described by Paxton
et al. (2007), Klee et al. (2007) and Chen et al. (2008).
Little is known about the epidemiology of
N. ceranae. Higes et al. (2008) demonstrated that N. ceranae can contaminate both honey bee pollen baskets and pollen collected from commercial apiarists. However, there are no reports of honey contaminated with N.
cer-anae. In Australia honey is commonly moved
from state to state, with the exception of honey destined for Western Australia. In this study, 37 honey samples sourced from Aus-tralia and overseas were tested for both N.
cer-anae and N. apis. Three samples were positive
for N. ceranae and eight samples were posi-tive for N. apis. The three honey samples that were positive for N. ceranae originated from Queensland where the prevalence of N.
122 T. Giersch et al.
detectable spores in these honey samples sup-ports the higher sensitivity of PCR compared to microscopy, but could also be an indication that there was non-spore associated Nosema DNA in honey.
Following the first detection of European foulbrood in Australia in 1977 (Tham,1978), Western Australia implemented restrictions on the importation of bees and bee products. Whilst European foulbrood has spread among the remaining states, Western Australia re-mains free of this disease. It seems likely that importation restrictions may have prevented
N. ceranae from entering Western Australia,
given that all 24 adult bee samples from West-ern Australia were N. ceranae negative when analysed by PCR-RFLP during this study. Nevertheless, a more comprehensive testing regime is required to unequivocally confirm that N. ceranae does not occur in Western Australia.
Tasmania is the state with the least number of beekeepers and colonies in Australia. It is also separated from mainland Australia by a large body of water. None of the 36 samples submitted from this state contained N.
cer-anae. Nevertheless, beekeepers in Tasmania
source queen bees from mainland Australia, so the importation of infected queens and escorts poses a risk for the introduction and establish-ment of N. ceranae in Tasmania.
Martin-Hernandez et al. (2007) carried out a study of bee samples collected in Spain from 1999 to 2005. Data from the first period 1999 to 2000 showed a consistency with the sea-sonal pattern usually associated with nosemo-sis, ie. an infection peak in spring (Langridge,
1961). However, in 2004 and 2005 the Nosema seasonality diminished to the point where, in 2005, consistently high numbers of Nosema-positive samples were detected throughout the year. In this study, 7 samples collected in March-April (Autumn), a time when Nosema is not commonly found, were confirmed to be
N. ceranae-positive, indicating that a similar
situation may be emerging in Australia. This study has confirmed that bees in Aus-tralia are infected with N. ceranae and that the prevalence of infection varies between states. Tasmania and Western Australia appear to be
N. ceranae free, but more intensive studies are
required to establish their freedom from this parasite. It was recently shown that pollen can be contaminated with N. ceranae (Higes et al.,
2008), and this study has demonstrated that both N. ceranae and N. apis can contaminate honey. As N. apis spores can stay viable in honey for several months depending on the honey type (Malone et al., 2001) it is likely that N. ceranae was introduced into Australia through imports of either contaminated honey or pollen. The extent of infected samples de-tected in multiple states indicates that N.
cer-anae has been in Australia for some time and
that any attempts at eradication would be un-likely to be successful.
ACKNOWLEDGEMENTS
We thank the Rural Industries Research & De-velopment Corporation for funding. We thank Pro-fessor Ingemar Fries from the Swedish University of Agricultural Sciences, Uppsala, Sweden for pro-viding a sample of Nosema ceranae spores which was used as the positive control for the molecular studies. We also thank the beekeepers, apiary o ffi-cers and Capilano Honey for submitting adult bee and honey samples.
Nosema ceranae infecte les abeilles
domes-tiques (Apis mellifera) et contamine le miel en Australie.
Nosema ceranae/ Nosema apis / nosémose /
pa-rasite/ Apis mellifera / détection
Zusammenfassung – Nosema ceranae infiziert Honigbienen (Apis mellifera) und kontaminiert Honig in Australien. Nosemosis ist die am
wei-testen verbreitete Krankheit der adulten Bienen und verursacht bei den Imkern weltweit erhebliche wirt-schaftliche Schäden. Im Jahr 1994 wurde bei der östlichen Honigbiene (Apis cerana) in China ein ähnliches Mikrosporidium wie N. apis beschrieben (Fries et al.,1996). Dieser Parasit, Nosema ceranae, wurde anschließend in europäischen Honigbienen (Apis mellifera) in Taiwan gefunden (Huang et al., 2005). In jüngerer Zeit wurde er in Brasilien, den USA, Vietnam und in mehreren europäischen Staa-ten nachgewiesen (Klee et al.,2007). Während bei
N. apis-Infektionen die Syndrome meist rasch
auf-treten und von begrenzter Dauer sind, verursacht
N. ceranae eher unspezifische Krankheitssymptome
ceranae kein neuer Parasit bei europäischen
Honig-bienen ist. So wurde N. ceranae in Bienenproben aus dem Jahr 1996 in den USA und 2002 in Frank-reich nachgewiesen (Chen et al., 2008; Chauzat et al.,2007). Die weit verbreiteten Nachweise von
N. ceranae sind eine Folge der molekularen
Nach-weismethoden, mit denen im Gegensatz zur mikro-skopischen Analyse eine klare Unterscheidung zwi-schen N. ceranae und N. apis möglich ist. Da es bisher keine Informationen bezüglich des Vorkom-mens von N. ceranae in Australien gibt, sollte mit dieser Studie geklärt werden, ob Honigbienen in Australien mit N. ceranae infiziert sind.
307 adulte Bienen und 37 Honigproben aus Austra-lien wurden mit PCR, RFLP (Restriction Fragment Length Polymorphism) und über mikroskopische Analysen auf das Vorkommen von N. ceranae und
N. apis hin untersucht. N. ceranae wurde in
Pro-ben aus 4 Staaten nachgewiesen: Queensland, New South Wales, Victoria und South Australia. Nicht nachgewiesen wurde N. ceranae in Proben aus We-stern Australia und Tasmania. N. apis wurde da-gegen in Proben aller Staaten gefunden. Drei Ho-nigproben von Imkern in Queensland waren für
N. ceranae positiv. Sechs Proben von
Importhoni-gen waren für beiden Nosema-Arten negative. Die-se Studie hat bestätigt, dass auch Bienen in Au-stralien mit N. ceranae infiziert sind und dass die Verbreitung der Infektion in den einzelnen Staaten unterschiedlich ist. Tasmania und Western Austra-lia scheinen noch frei von N. ceranae zu sein, doch müsste dies mit umfangreicheren Analysen abgesi-chert werden. Der Umfang der positiven Nachwei-se in mehreren Staaten weist darauf hin, dass N.
ceranae bereits seit einiger Zeit in Australien
vor-kommt und dass Versuche zur Ausrottung des Erre-gers kaum Aussicht auf Erfolg haben.
Nosema ceranae/ Nosema apis / Nosemosis / Apis mellifera/ PCR / RFLP
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